Systems for gene targeting and producing stable genomic transgene insertions

ABSTRACT

The novel germ-line transformation systems disclosed in this patent application allow the physical deletion of transposon DNA following the transformation process, and the targeting of transgene integrations into predefined target sites. In this way, transposase-mediated mobilization of genes-of-interest is excluded mechanistically and random genomic integrations eliminated. In contrast to conventional germ-line transformation technology, our systems provide enhanced stability to the transgene insertion. Furthermore, DNA sequences required for the transgene modification (e.g. transformation marker genes, transposase or recombinase target sites), are largely removed from the genome after the final transgene insertion, thereby eliminating the possibility for instability generated by these processes. The RMCE technology, which is disclosed in this patent application for invertebrate organisms (exemplified in  Drosophila melanogaster ) represents an extremely versatile tool with application potential far beyond the goal of transgene immobilization. RMCE makes possible the targeted integration of DNA cassettes into a specific genomic loci that are pre-defined by the integration of the RMCE acceptor plasmid. The loci can be characterized prior to a targeting experiment allowing optimal integration sites to be pre-selected for specific applications, and allowing selection of host strains with optimal fitness. In addition, multiple cassette exchange reactions can be performed in a repetitive way where an acceptor cassette can be repetitively exchanged by multiple donor cassettes. In this way several different transgenes can be placed precisely at the same genomic locus, allowing, for the first time, the ability to eliminate genomic positional effects and to comparatively study the biological effects of different transgenes.

FIELD OF THE INVENTION

The invention relates to novel methods and techniques to producetransgenic, or genetically modified, organisms (transgenesis). The focusof the innovation is on manipulation techniques that allow for thetargeting and the stable anchoring of homologous or heterologousDNA-sequences (in the following description referred to as: “transgene”or “gene-of-interest”) into the genome of a target species. To achievethis goal, we have developed three different systems of transformationvectors that are capable of integrating a transgene into invertebrateand vertebrate organisms via transposon- or recombinase-mediatedtransformation events. In addition, following the germlinetransformation procedure, both systems make possible the physicaldeletion of mobile DNA-sequences, brought in with the vector, from thetarget genome and therefore to stabilize the gene-of-interest. Stable(genomic) transgene insertions are regarded to be an essentialpre-requisite for the safe production of genetically modified organismsat a large industrial scale.

DESCRIPTION OF THE RELATED ART

Current state-of-the-art technology to produce genetically modifiedinsect organisms relies on transposon-mediated germ-line transformation.This transformational technique is based on mobilizable DNA, i.e.transformation vectors derived from Class II transposable elementshaving terminal inverted sequences, which transpose via a DNA-mediatedprocess (see Finnegan, D. J., 1989. Eucaryotic transposable elements andgenome evolution. Trends Genet. 5, 103-107, and Atkinson, P. W.,Pinkerton, A. C., O'Brochta, D. A., 2001. Genetic transformation systemsin insects. Annu. Rev. Entomol. 46, 317-346, the contents of which areincorporated herein by reference). The two ends of such a transposableelement carrying within all functional parts necessary and sufficientfor in vivo mobilization are termed TransposonL (5′ end) and TransposonR(3′ end) Several different germ-line transformation systems have incommon that a gene-of-interest/transgene originally located within atransgene construct is transferred into genomic DNA of germ-line cellsof the target species. The transformation process is catalyzed by thetransposase enzyme provided by a helper plasmid. This enzyme recognizesDNA target sites flanking the gene-of-interest/transgene and mobilizesthe transgene into the genome of germ-line cells of the insect species.In addition, transformed DNA contains a marker gene that allowsdetection of successful germ-line transformation events (by producing adominantly visible phenotype).

Transposon-mediated germ-line transformation systems are currentlyavailable for a diverse spectrum of insect species. Systems based on theP-element revolutionized the genetics of the vinegar fly Drosophilamelanogaster (see Engels, W. R. (1996). P elements in Drosophila. Curr.Top. Microbiol. Immunol. 204, 103-123, the contents of which areincorporated herein by reference), but they were not applicable tonon-drosophilid insect species because of the dependence of P-elementson Drosophila-endogenous host factors (see Rio, D. C. & Rubin, G. M.(1988). Identification and purification of a Drosophila protein thatbinds to the terminal 31-base-pair inverted repeats of the Ptransposable element. Proc. Natl. Acad. Sci. USA 85, 8929-8933, thecontents of which are incorporated herein by reference). Therefore,insect species of medical or economic importance have been transformedusing host factor-independent “broad host range” transposable elements(see Atkinson, P. W. & James, A. A. (2002). Germline transformantsspreading out to many insect species. Adv. Genet. 47, 49-86, thecontents of which are incorporated herein by reference). Germlinetransformation systems based on the transposable elements piggyBac (seeU.S. Pat. No. 6,218,185; WO 01/14537; and Handler, A. M., McCombs, S.D., Fraser, M. J., Saul, S. H. (1998). The lepidopteran transposonvector, piggyBac, mediates germline transformation in the Mediterraneanfruitfly. Proc. Natl. Acad. Sci. USA 95, 7520-7525, the contents ofwhich are incorporated by reference herein), Hermes (see U.S. Pat. No.5,614,398, the contents of which are incorporated herein by reference),Minos (see European Patent No. EP 0 955 364 A36, the contents of whichare incorporated herein by reference) and mariner (see WO 99/09817, thecontents of which are incorporated herein by reference) are currentlystate-of-the-art technology to genetically modify important pest oruseful insect species including, for example, malaria transmittinganopheline or culicine mosquitoes (Anopheles gambiae, Anophelesstephensi, Anopheles albimanus, Culex quinquefasciatus, Aedes aegypti;see Catteruccia, F., Nolan, T., Loukeris, T. G., Blass, C., Savakis, C.,Kafatos, F. C. & Crisanti, A. (2000). Stable germline transformation ofthe malaria mosquito Anopheles stephensi. Nature 405, 959-962, andAllen, M. L., O'Brochta, D. A., Atkinson, P. W. & Levesque, C. S.(2001). Stable, germ-line transformation of Culex quinquefasciatus(Diptera: Culicidae). J. Med. Entomol. 38, 701-710, and Coates J. C.,Jasinskiene, N., Miyashiro, L. & James, A. A. (1998). Marinertransposition and transformation of the yellow fever mosquito, Aedesaegypti. Proc. Natl. Acad. Sci. USA 95, 3748-3751, and Jasinskiene, N.,Coates, C. J., Benedict, M. Q., Cornel, A. J., Rafferty, C. S., James,A. A. & Collins, F. H. (1998). Stable transformation of the yellow fevermosquito, Aedes aegypti, with the Hermes element from the housefly.Proc. Natl. Acad. Sci. USA 95, 3743-3747, and Perera, O. P., Harrell, R.A., Handler, A. M. (2002) Germ-line transformation of the South Americanmalaria vector, Anopheles albimanus, with a piggyBac/EGFP transposonvector is routine and highly efficient. Insect Mol. Biol., 11, 291-297,the contents of which are incorporated herein by reference), theMediterranean fruit fly, Ceratitis capitata (see Handler, A. M.,McCombs, S. D., Fraser, M. J., Saul, S. H. (1998). The lepidopterantransposon vector, piggyBac, mediates germline transformation in theMediterranean fruitfly. Proc. Natl. Acad. Sci. USA 95, 7520-7525 andLoukeris, G. T., Livadaras, I., Arca, B, Zabalou, S. & Savakis, C.(1995). Gene transfer into the Medfly, Ceratitis capitata, with aDrosophila hydei transposable element. Science 270, 2002-2005, thecontents of which are incorporated herein by reference) and thesilkworm, Bombyx mori (see Tamura, T. et al. (2000). Germlinetransformation of the silkworm Bombyx mori L. using a piggyBactransposon-derived vector. Nat. Biotechnol. 18, 81-84, the contents ofwhich are incorporated herein by reference). Moreover, the applicationpotential of broad host range transposable elements is not restricted toinsect species: mariner-derived transformation vectors have been shownto integrate stably into the germ-line of the nematode, Caenorhabditiselegans (see Bessereau, J.-L., Wright, A., Williams, D. C., Schuske, K.,Davis, M. W. & Jorgensen, E. M. (2001). Mobilization of a Drosophilatransposon in the Caenorhabditis elegans germ line. Nature 413, 70-74,the contents of which are incorporated herein by reference), thezebrafish, Danio rerio (see Fadool J. M., Hartl, D. L. & Dowling, J. E;(1998). Transposition of the mariner element from Drosophila mauritianain Zebrafish. Proc. Natl. Acad. Sci. USA 95, 5182-5186, the contents ofwhich are incorporated herein by reference) and chicken, Gallus spp.(see Sherman, A., Dawson, A., Mather, C., Gilhooley, H., Li, Y.,Mitchell, R., Finnegan, D. & Sang, H. (1998). Transposition of theDrosophila element mariner into the chicken germ line. Nat. Biotechnol.16, 1050-1053, the contents of which are incorporated herein byreference).

In order to follow germ-line transformation success, bothspecies-specific and species-independent transformation markers havebeen established (see Horn, C., Schmid, B. G. M., Pogoda, F. S. &Wimmer, E. A. (2002). Fluorescent transformation markers for insecttransgenesis. Insect Biochem. Mol. Biol. 32, 1221-1235, the contents ofwhich are incorporated herein by reference). Species-independent markersconsist of a combination of a promoter sequence which isphylogenetically conserved and a gene for a fluorescent protein placedunder control of such a promoter (for example, GFP [green fluorescingprotein] and derivatives thereof, or DsRed [Discosoma species redfluorescing protein] (see Chalfie, M. Tu, Y., Euskirchen, G., Ward, W.,Prasher, D. C. (1994). Green fluorescent protein as a marker for geneexpression. Science 263, 802-805, and Matz, M. V., Fradkov, A. F.,Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L.,Lukyanov, S. A. (1999). Fluorescent proteins from nonbioluminescentAnthozoa species. Nat. Biotechnol. 17: 969-973, the contents of whichare incorporated herein by reference). Species-independent markers areadvantageous over species-specific markers because they are directlyapplicable to different insect species (and other organisms). Thepolyubiquitin-promoter (see Patent Cooperation Treaty PCT WO 01/14537 A1and Handler, A. M. & Harrell, R. A. (1999). Germline transformation ofDrosophila melanogaster with the piggyBac transposon vector. Insect Mol.Biol. 8, 449-457, the contents of which are incorporated herein byreference) as well as the “3×P3”-promoter (see Patent Cooperation TreatyPCT WO 01/12667 A1 and Berghammer, A. J., Klingler, M., & Wimmer, E. A.(1999). A universal marker for transgenic insects. Nature 402, 370-371,the contents of which are incorporated herein by reference) linked togenes for fluorescent proteins have been used most widely for thispurpose.

A transposon-independent technology aiming at targeting agene-of-interest/transgene into the genome of cells relies on theprinciple of site-specific recombination. This is possible by using arecombinase enzyme and corresponding DNA target sites that areheterospecific. The steps are: First, incorporating into the genome bytransposon-mediated transformation, a DNA cassette that is flanked byheterospecific recombinase target sites and contains a marker system forpositive-negative selection. Second, recombinase-mediated targeting intothe marked genomic locus the gene-of-interest, which is located within aplasmid and is flanked by the same heterospecific recombinase targetsites. This principle has been described as RMCE or recombinase-mediatedcassette exchange (see European Patent No. EP 0 939 120 A1 and Baer, A.& Bode, J. (2001). Coping with kinetic and thermodynamic barriers: RMCE,an efficient strategy for the targeted integration of transgenes. CurrOpin Biotechnol. 12, 473-480 and Kolb, A. F. (2002). Genome engineeringusing site-specific recombinases. Cloning Stem Cells. 4, 65-80, thecontents of which are incorporated herein by reference). Thefunctionality of DNA cassette exchange systems has been demonstrated indifferent cell lines (comprising also murine embryonic stem cells) usingthe FLP-recombinase enzyme and heterospecific FRT target sites (seeSchlake, T. & Bode, J. (1994). Use of mutated FLP recognition target(FRT) sites for the exchange of expression cassettes at definedchromosomal loci. Biochemistry 33, 12746-12751, and Seibler, J.,Schübeler, D., Fiering, S, Groudine, M. & Bode, J. (1998). DNA cassetteexchange in ES cells mediated by Flp recombinase: an efficient strategyfor repeated modification of tagged loci by marker-free constructs.Biochemistry 37, 6229-6234, and European Patent No. EP 0 939 120 A1, thecontents of which are incorporated herein by reference) as well as usingthe Cre-recombinase enzyme and heterospecific loxP target sites (seeKolb, A. F. (2001). Selection-marker-free modification of the murinebeta-casein gene using a lox2272 [correction of lox2722] site. AnalBiochem. 290, 260-271.26), the contents of which are incorporated hereinby reference). However, RMCE has not been applied to geneticallymodified invertebrate organisms thus far.

Limitations of Prior Art/Improvements Over Prior Art

Transposon-based plasmid vectors have proven to be efficient tools forproducing genetically modified insects for research purposes, but so faronly on a small laboratory scale. However, the mobile nature of DNAtransposable elements will be disadvantageous when scaling up theproduction/rearing of genetically modified insects. Owing to potentialre-mobilization, the stability of genomic transgene integrations cannotbe assured and, connected to this issue, concerns relating to the safetyof release of such genetically modified insects will be raised.

Stability of Genomic Transgene Integrations in Large Industrial Scale

The current state-of-the-art provides, typically, for random transposonvector integrations into the host genome. While this may be advantageousfor functional genomics studies that use vector integrations to causerandom mutations (e.g. for transposon-tagging and enhancer trapping), itis typically disadvantageous for the creation of transgenic strains forapplied use where high fitness levels and optimal transgene expressionare desired. This results from integrations that create mutations byinsertion into genomic sites that eliminate or disrupt normal genefunction that negatively effect viability, reproduction, or behavior.Genomic position effects also influence expression of transgenes,typically causing decreased expression and/or mis-expression of genes ofinterest and markers so that transformants may not be easily identified,and the desired transgene expression for application is not achieved.Thus, most transformation experiments require the screening of multipletransformant strains for optimal fitness and transgene expression, andoften such strains cannot be identified. An important improvement overthe current state-of-the-art would be an efficient and routine system totarget transgene integrations to specific and defined genomic sites thatare known not to disrupt normal gene function and whose position effectsare limited or well characterized.

Transgene integrations that negatively effect host strain fitness andreproduction also confer a selective disadvantage to the transformedorganism in a population relative to wild type organisms. Thus, aselective advantage is provided to non-transformed organisms ortransformants that have lost or relocated the transgene due to are-mobilization event. Re-mobilization requires the activity of atransposase enzyme corresponding to, and acting upon, the transposonsequences flanking the genomic transgene. Although the transposase usedfor germ-line transformation usually is not encoded by the host species'genome, transposase introduction by symbiotic or infectious agents ispossible, and cross-reactivity to related transposase enzymes that aregenomically encoded cannot be excluded. Such cross-reactivities havebeen reported between the transposable elements Hermes, from Muscadomestica, and hobo, from Drosophila melanogaster, that causedsignificant instability of Hermes-flanked transgenes in hobo-containingDrosophila strains (see Sundararajan, P., Atkinson, P. W. & O'Brochta,D. A. (1999). Transposable element interactions in insects:crossmobilization of hobo and Hermes. Insect Mol. Biol. 8, 359-368, thecontents of which are incorporated herein by reference). It should benoted that well-characterized families of transposable elements containmultiple members and the cross-reactivity of them is largely unknown todate (e.g. the mariner/Tc1 superfamily (see Hartl, D. L., Lohe, A. R. &Lozovskaya, E. R. (1997). Modern thoughts on an ancyent marinere:function, evolution, regulation. Annu. Rev. Genet. 31, 337-358, thecontents of which are incorporated herein by reference)). For thesereasons, a transformation technology that excludes the possibility oftransgene re-mobilization events a priori will provide a higher standardof transgene stability and will be superior to currently availabletechnology.

Transgene instability resulting from vector remobilization will haveseveral negative consequences. The first is loss or change in desiredtransgene expression. Secondly, strain breakdown will result afterrelocated transgenes can segregate freely in meiosis and selectionpressure acts against transgene-carrying chromosomes. Research resultson the stability of transgene insertions in insects, reared at anindustrial scale, have not been reported thus far. However, data forinsect strains selected by classical Mendelian genetics and carryingtranslocations are available (see Franz, G., Gencheva, E. & Kerremans,Ph. (1994). Improved stability of genetic sex-separation strains for theMediterranean fruit fly, Ceratitis capitata. Genome 37, 72-82, thecontents of which are incorporated herein by reference). When reared atan industrial scale, such translocation strains, constructed for theMediterranean fruit fly (see Franz, G., Gencheva, E. & Kerremans, Ph.(1994). Improved stability of genetic sex-separation strains for theMediterranean fruit fly, Ceratitis capitata. Genome 37, 72-82, thecontents of which are incorporated herein by reference) suffered frominstability. Recombination events causing reversion of the selectedrecessive trait were observed at a frequency of 10⁻³-10⁻⁴ (see Franz, G.(2002). Recombination between homologous autosomes in medfly (Ceratitiscapitata) males: type-1 recombination and the implications for thestability of genetic sexing strains. Genetica 116, 73-84, the contentsof which are incorporated herein by reference). Because the recessivetrait conferred a selective disadvantage to the individual insect, suchreversion events caused strain breakdown rapidly. Most interestingly,these events were not observed at a small laboratory scale and thereforewere not anticipated. As strain breakdown during a continuous industrialproduction of those insects is not acceptable, major research effortshave been made to improve the situation. Currently a laborious (andexpensive) but efficient manual detection system for quality control hasbeen implemented (see Fisher, K. & Caceres, C. (2000). A filter rearingsystem for mass reared medfly, S. 543-550 in Area-wide control of fruitflies and other insect pests, Ed.: Tan, K. H., Penerbit Universiti SainsMalaysia, Penang, Malaysia, the contents of which are incorporatedherein by reference) and allows the successful production of thistranslocation strain at a scale of 10⁶-10⁷ individuals per week (seeFranz, G. (2002). Recombination between homologous autosomes in medfly(Ceratitis capitata) males: type-1 recombination and the implicationsfor the stability of genetic sexing strains. Genetica 116, 73-84, thecontents of which are incorporated herein by reference).

Safety Aspects Concerning Release of Genetically Modified Insects

Another important concern for remobilization is the potential forlateral transmission of the transgene into unintended host strains orspecies. Many industrial applications of insect transgene technologywill include the release of genetically modified insects into theenvironment (e.g. the Sterile Insect Technique). Therefore, aspects ofbiosafety and ecological risk assessment will be of fundamentalimportance. Biosafety includes minimizing the risk of unintendedtransgene transmission from the host to other procaryotic or eucaryoticspecies during rearing or after release into the field. Horizontal genetransfer cannot be excluded per se, because the mechanisms of nucleicacid exchange between species are not sufficiently investigated to date.While most transposon vectors have their transposase source eliminatedand are not self-mobilizable, functional autonomous transposons can betransmitted among species horizontally, and transposase may be providedto the vector by associated organisms or by a related enzyme in the hostspecies. Thus, the risk for transgene vector re-mobilization by atransposase-mediated event can be most definitively eliminated whentransposon sequences, required for germ-line transformation, are removedfrom the genomic integration after the transformation process. Systemsdisclosed in this patent application contribute to risk minimization byintroducing techniques for transposon sequence removal. It is probablethat, in the future, procedures to remove such sequences, and thereforeto assure a higher standard of biosafety, will become an obligateprecondition for permission by regulatory organizations for release oftransgenic organisms. In fact, there are sound prospects that suchsystems will set the safety standards and will become normative which inturn demonstrates the commercial potential of the invention.

BRIEF SUMMARY OF THE INVENTION The Strategy Post-TransformationalImmobilization of Transgenes

Disadvantages stated in the previous section show the need for novelgerm-line transformation systems that enable the stable integration oftransgenes/genes-of-interest. The challenge is to develop atransformation method that prevents re-mobilization of transgenes whichhave been incorporated into the genome. The strategy disclosed in thispatent application is to remove the intact transposon parts (containingtransposase-recognition sites) following the transformation procedure(i.e. post-transformational). Three variants of this invention aredisclosed as embodiments. These variants allow (i) modification oftransgene DNA, (ii) post-transformational inactivation of at least oneof the transposon parts and (iii) inactivation of at least one of thetwo transposon recognition sites required for re-mobilization byphysical deletion from the genome.

The first embodiment disclosed has been termed “excision-competentstabilization vectors” (FIG. 1). This embodiment comprises atransformation vector that, in addition to currently applied vectorsthat contain solely a TransposonL1 half side and TransposonR1 half side(now referred to as TransposonL1 and R1), contains an additionalinternally-positioned TransposonL half side (referred to as TransposonL2in FIG. 1) placed in-between the original Transposon L1 and R1 sides. Land R half sides are placed in the normal, or same, terminal invertedrepeat orientation to one another as found in the original transposableelement. Marker genes that can be distinguished from one another areplaced in-between TransposonL1 and TransposonL2 and in-betweenTransposonL2 and TransposonR1. The steps of transformation are asfollows. First, the transformation procedure is carried out according tothe current state-of-the-art germ-line transformation technology thatwill result in individuals transformed by one of two possible eventswith this vector. One possible event is the integration of TransposonL1and TransposonR1 and all intervening DNA including the two marker genes,TransposonL2, and other genes of interest. The second possible event isintegration of TransposonL2 and TransposonR1 and all intervening DNAincluding the marker gene. For the purposes of this embodiment, onlyindividuals transformed with TransposonL1 and TransposonR1, which areidentified by expression of the two marker genes, are conserved forfurther experimentation. The internal vector containing TransposonL2 andTransposonR1, within TransposonL1 and TransposonR1, is then re-mobilizedby introduction of a source of transposase derived from mating to ajumpstarter strain having a genomic transposase gene, or physicalinjection of the transposase DNA, RNA, or protein into embryos. Deletionby transposon excision of the TransposonL2 and TransposonR1 half sidesis identified by loss of the intervening marker gene. The remainingTransposonL1 half-side, with the downstream marker gene andgenes-of-interest, is identified by the single marker gene phenotype andverified by sequencing of amplified DNA. This remaining TransposonL1half side, marker gene and genes-of-interest should be incapable ofre-mobilization by transposase in the absence of the requisiteTransposonR1 half side.

The second embodiment disclosed has been termed “conditionalexcision-competent transformation vectors” (FIG. 4). This embodimentcomprises a modified excision-competent transformation vector thatcontains a transposonR2 half-side in an inverted orientation, relativeto the R1 half side, with R2 also flanked by recombinase target sites ininverted orientation. In this configuration, only the TransposonL1 andR1 half-sides can integrate by transposition, and remobilization of theTransposonL1 and R2 half-sides can only occur after arecombinase-mediated inversion between the recombinase target sites.This modification will facilitate the stabilization process, bytransposon L1 and R2 half-side deletion, for those excision-competenttransformation vectors and/or host species where the primarytransposition is highly favored or limited to the internal TransposonL1and R2 half-sides if R2 was in a normal orientation.

A similar result is achieved by the third embodiment which has beentermed “RMCE with subsequent transposon deletion” (FIG. 5). Completelynew in this embodiment is a DNA targeting strategy. The ultimategerm-line transformation process is conducted as a recombinase-mediatedprocess, instead of a transposase-mediated process, into an existing(and pre-defined) genomic target site. This involves the RMCE principle,i.e. a site-specific recombinase recognizes heterospecific DNA targetsites and exchanges DNA-cassettes between a RMCE-acceptor and aRMCE-donor (step 1 in FIG. 5). The success of this cassette exchange isindicated by the exchange of the acceptor target marker gene (e.g. ECFP,see FIG. 7) by the donor vector marker gene (e.g. EYFP, see FIG. 7). Itis important to stress that only the coding region of the transformationmarket genes is exchanged, not the promotor regions (which are notpresent in the RMCE-donor plasmid). The advantage of this promoter-freeexchange is that side-reactions, which involve non-targeted integrationof the donor into the genome, will not be recognized. Most important tothis first step of cassette exchange, is a “homing DNA sequence” that ispresent in both the RMCE-acceptor and the RMCE-donor and is identical inboth functional parts. The homing DNA sequence functions tosignificantly enhance the cassette exchange efficiency. The principle ofstably integrating a gene-of-interest via a RMCE strategy into thegenome of an invertebrate organism is completely novel and extendspreviously described RMCE-technology (see European Patent No. EP 0 939120 and Schlake, T. & Bode, J. (1994). Use of mutated FLP recognitiontarget (FRT) sites for the exchange of expression cassettes at definedchromosomal loci. Biochemistry 33, 12746-12751, and Seibler, J.,Schübeler, D., Fiering, S, Groudine, M. & Bode, J. (1998). DNA cassetteexchange in ES cells mediated by Flp recombinase: an efficient strategyfor repeated modification of tagged loci by marker-free constructs.Biochemistry 37, 6229-6234, and European Patent No. EP 0 939 120, thecontents of which are incorporated herein by reference) to invertebrateorganisms. Because the RMCE-acceptor also carries a transposon half-side(Transposon R1 in FIG. 5), a fully remobilizable internal transposon isreconstituted after a successful RMCE reaction. This reconstitutedtransposon is subsequently physically deleted from the organism's genomeby the action of a transposase (step 2 in FIG. 5 and FIG. 7), exactly asdescribed for the first embodiment. In conclusion, the gene-of-interestis only flanked by one transposon half side end and hence is immobilizedbecause it does not provide a complete substrate fortransposase-mediated mobilization.

BRIEF DESCRIPTION OF THE FIGURES

For a fuller understanding of the nature and objects of the presentinvention, reference should be made by the following detaileddescription taken with the accompanying figures, in which:

FIG. 1 shows a protocol for integration and re-mobilization forstabilized vector creation;

FIG. 2 shows a diagram of stabilization vectorpBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1 };

FIG. 3 shows a PCR analysis and verification ofpBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} vector integration in line F34 andL2-3×P3-ECFP-R1 remobilization in line F34-1M;

FIG. 4 shows the principle of “conditional excision competenttransformation vectors”;

FIG. 5 shows the principle of “RMCE with subsequent transposondeletion”;

FIG. 6 shows an embodiment of the principle as shown in FIG. 4

FIG. 7 shows an embodiment of the principle as shown in FIG. 5:Stabilized vector creation by RMCE;

FIG. 8 shows a diagram of RMCE acceptor vector;

FIG. 9 shows molecular analysis of RMCE acceptor and RMCE donortransgenic lines and PCR analysis of transgene mobilization;

FIG. 10 shows a diagram of a final RMCE donor vector for transgenestabilization;

FIG. 11 shows the approximate sequence of the vector shown in FIG. 2;

FIG. 12 shows the approximate sequence of the vector shown in FIG. 8;and

FIG. 13 shows the approximate sequence of the vector shown in FIG. 10.

DETAILED DESCRIPTION OF THE INVENTION Embodiment 1 Excision-CompetentStabilization Vectors

The experimental steps for the method are described in FIG. 1, and thestructure of the excision competent transformation vector,pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1 }, is described in FIG. 2. Integrationand re-mobilization of the vector was verified by PCR and sequenceanalysis described in FIG. 3. pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} wasconstructed based on the transposable element “piggyBac” (see U.S. Pat.No. 6,218,185, the contents of which are incorporated herein byreference). Conventional piggyBac-based transformation vectors (see WO01/14537 and WO 01/12667, the contents of which are incorporated hereinby reference) typically contain piggyBac-half sides or parts thereof,including 5′ piggyBac terminal sequences (referred to as piggyBacL) and3′ piggyBac terminal sequences (referred to as piggyBacR), which flank atransformation marker gene and a cloning site to insert thegenes-of-interest. (see Handler, A. M., 2001. A current perspective oninsect gene transfer. Insect Biochem. Mol. Biol., 31, 111-128, thecontents of which are incorporated herein by reference.) For vectorsthat are not autonomously transpositionally active, the transposase geneis partially deleted or interrupted by marker genes orgenes-of-interest, thereby mutating the transposase. Non-autonomousvectors require an independent source of functional transposase formobilization resulting in transposition. In contrast to conventionalvectors, pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} is provided with anadditional piggyBacL half side (referred to as L2 half side) that is inthe same orientation as the L1 half side, and positioned internal to thepiggyBac L1 and R1 half sides. In this orientation, transposition canoccur utilizing the L1 and R1 half sides, or the internal L2 and R1 halfsides. In addition, pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} contains a uniqueKasI restriction endonuclease site in the piggyBacL1 region that can beused to insert genes of interest. In order to follow the primarytransformation integration event of the L1 and R1 half-sides and todistinguish it from integration of L2 and R1 half-sides, independenttransformation marker genes are placed in-between the two half-sidepairs. In pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1 }, the PUbDsRed1 (see WO01/14537, the contents of which are incorporated herein by reference)marker is placed in-between the L1 and L2 half sides, and the 3×P3-ECFP(see WO 01/12667, the contents of which are incorporated herein byreference) marker is placed in-between, the L2 and R1 half sides.

pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1}:

A 3.7-kb AflIII-AflII fragment from pB[PUbDsRed1], containing 0.7 kb ofpiggyBac L1 half-side DNA and adjacent 5′ insertion site DNA and thepolyubiquitin:DsRed1 DNA gene, was blunted by Klenow-mediated nucleotidefill-in reaction and isolated by agarose gel purification. The bluntedfragment was ligated into the MscI site of pXL-BacII-3×P3-ECFP. Plasmidshaving the 3×P3-ECFP and polyubiquitin:DsRed1 reading frames in oppositeorientation were selected.

phspBac Transposase Helper Plasmid:

For germline transformation experiments, the helper phspBac was (see PCTWO 01/14537, the contents of which are incorporated herein byreference).

Experimental Steps of the Transgene Immobilization Process:

a) Germ-Line Transformation with pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1 }

The pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} vector was integrated into theDrosophila genome of the white eye w[m] strain by coinjection with thephspBac helper plasmid into pre-blastoderm embryos. Using conventionalpiggyBac-mediated germ-line transformation methods (see U.S. Pat. No.6,218,185 and WO 01/14537, the contents of which are incorporated hereinby reference), seven putative G1 transformant lines expressing only the3×P3-ECFP marker were observed and discarded. One G1 male fly exhibitedboth thoracic expression of DsRed and eye expression of ECFP, and it wasbackcrossed to w[m] females to create a line designated as F34.Transformation by an intact pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} vector bypiggyBac-mediated transformation in F34 was confirmed by sequencing ofinternal PCR products and inverse PCR products, derived from F34 genomicDNA, which provided the insertion site DNA sequence (see below).

b) PiggyBac Transposase-Induced Excision of PiggyBacL2 and PiggyBacR1

Transformed individuals identified and confirmed to have the markergenes 3×P3-ECFP and PUbDsRed1 were backcrossed to w[m] flies for twogenerations. The presence of both markers solely in female progeny fromF34 parental males indicated X-chromosome sex-linkage for the primaryintegration. F34 flies were mated as transgene heterozygotes to apiggyBac jumpstarter strain (w+/Y;pBac/pBac;+/+) having a homozygousP-element-mediated integration of an hsp70-regulated piggyBactransposase gene into chromosome 2 and marked with the wild type white+allele. Larval and pupal offspring of these matings were heat shocked at37° C. for 60 minutes every second day until adult emergence to promotetransposase gene expression. Male and female progeny of these matingswere screened, with those carrying the transposase gene (red eyepigmentation) and expressing the fluorescent protein markers, PUb-DsRed1and 3×P3-ECFP, being mated to w[m] individuals. Ten matings of 4 to 5appropriately marked females to w[m] males and 18 matings of 2 to 3marked males to w[m] females were set up. Progeny from these matingswere screened for expression of PUb-DsRed1 and the absence of 3×P3-ECFP,which would indicate loss by remobilization of the piggyBacL2 andpiggyBacR1 half sides with the intervening 3×P3-ECFP marker DNA. Progenyexpressing only DsRed1 fluorescence were detected at an approximatefrequency of 2% of all flies screened. A single white eye male (lackingthe transposase gene) and expressing DsRed1, and not ECFP, wasoutcrossed to w[m] females with the resultant line designated as F34-1M.

c) Molecular Analysis of the Vector Integration Before and afterRemobilization

The pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} integration into the F34Drosophila genome was initially identified by phenotypic expression ofthe DsRed and ECFP marker genes and verified by PCR amplication oftransformant DNA using primers internal to the vector sequence (see FIG.3). Genomic insertion site DNA flanking the integration was obtained byinverse PCR of the piggyBacL1 5′-end half side using the 122R and 139Fprimers, in outward orientation, to F34 genomic DNA digested with MspIendonuclease and circularized by ligation. The 5′ end insertion sitesequence was compared by BLAST analysis to the Drosophila GenomeSequence Database, and consistent with segregation analysis, was foundto be homologous to sequence found on the X-chromosome at locus 9B4. Thedatabase sequence was used to derive the piggyBacR1 3′-end insertionsite, and the 197F and 196R PCR primers were created to genomicinsertion site DNA at the 5′ and 3′-end flanking sequences,respectively. The genomic primers were then used to amplify and sequenceDNA that spans the pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} integration inF34, to further verify it as a primary intact piggyBac vectorintegration. The 197F and 196R primers were then used for PCR of F34-1Mgenomic DNA, which confirmed remobilization of the L2-PUbDsRed1-R1internal vector DNA in F34. Further verification of the vectorintegration and subsequent re-mobilization was achieved by sequencing ofPCR products obtained with primers 196 and 197 in combination withprimers to internal vector DNA described in FIG. 3. In all cases,positive PCR results yielded sequences consistent with a primaryintegration of pBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1} in F34, andremobilization of the L2-PUbDsRed1-R1 sequence in F34-1M flies. PCRproducts were not obtained in F34-1M flies using primers to theL2-PUbDsRed1-R1 sequence consistent with its deletion from the genomicDNA after re-mobilization.

Embodiment 2 Conditional Excision-Competent Transformation Vectors

The structure of the conditional excision-competent transformationvector, pBac_STBL, as well as the experimental steps are depictedschematically in FIGS. 4 and 6. pBac_STBL is based on the transposableelement “piggybac” (see U.S. Pat. No. 6,218,185, the contents of whichare incorporated herein by reference) and is a modified version ofpBac{L1-PUbDsRed1-L2-3×P3-ECFP-R1 }. In pBac-STBL the internaltransposon half-side (R2) is a duplication of the piggyBac 3′-end, andit is in reverse, or opposite, orientation to R1. In addition, it isflanked in upstream and downstream positions by FRT (FLP recombinasetarget) sites in opposite directions that create an inversion byrecombination in the presence of FLP recombinase (see FIGS. 4 and 6).Therefore, in this vector, only the piggyBacL1 and R1 half sides andintervening DNA can integrate, but re-mobilization of piggyBacR2together with piggyBacL1 or piggyBacR1 should not be possible.Mobilization of piggyBacR2 and L1 is only possible after FRTrecombination.

In addition, pBac_STBL contains unique cloning sites for the rareoctamer-specific restriction enzymes AscI and FseI. pBac_STBL isequipped with two separable transformation marker genes (see WO01/12667, the contents of which are incorporated herein by reference),which are located upstream of the AscI/FseI cloning sites (3×P3-EYFP;FIG. 6) and downstream of the FRT-sites (3×P3-DsRed; FIG. 6),respectively. In the following, the details of pBac_STBL plasmidconstruction starting from plasmid vectors already published aredisclosed:

pSL-3×P3-DsRedaf:

A 0.8 kb SalI-NotI fragment from pDsRed1-1 (Clontech, Palo Alto, Calif.)is cloned into the plasmid pSL-3×P3-EGFPaf (see WO 01/12667, thecontents of which are incorporated herein by reference) previouslydigested with SalI-NotI. Thereby, the EGFP (0.7 kb) open reading framewas replaced by the DsRed (0.8 kb) open reading frame.

pSLfaFRTfa:

The FRT sequence (90 bp) is prepared by SalI-Asp718 restriction ofPSL>AB> and cloned into the plasmid pSLfa1180fa previously digested withXhoI-Asp718. The FRT sequence corresponds to the substrate of the FLPrecombinase:

TT GAAGTTCCTATTC C GAAGTTCCTATTC TCTAGAAA GTATAGGAACTTCAGAGCGCTTTTGAAGCTpSL-3×P3-DsRed-FRT:

A 1.0 kb EcoRI-BsiWI fragment from pSL-3×P3-DsRedaf (containing theDsRed-ORF under 3×P3 promoter control) is cloned into pSLfaFRTfapreviously digested with EcoRI-Asp718.

pSL-3×P3-DsRed-FRT-FRT:

The PCR amplification product of the FRT sequence (template: pSL>AB>;Primers: CH_FRT_F 5′-GAGCTTAAGGGTACCCGGGGATCTTG-3′ and CH_FRT_R

5′-GACTAGTCGATATCTAGGGCCGCCTAGCTTC-3′) is digested with BfrI-SpeI andcloned into pSL-3×P3-DsRed-FRT previously digested with BfrI-SpeI. BothFRT sequences are oriented in opposite directions.

pSL-3×P3-DsRed-FRT-pBacR2-FRT:

The piggyBac 3′ sequence (referred to as: piggyBacR2) is prepared as a1.3 kb HpaI-EcoRV fragment from the plasmid p3E1.2 (see U.S. Pat. No.6,218,185, the contents of which are incorporated herein by reference)and cloned into the plasmid pSL-3×P3-DsRed-FRT-FRT previously cut withEcoRV. The piggyBacR2 insertion with an orientation opposite to theDsRed-ORF is chosen (the EcoRV cloning site is restored at the 5′ end ofthe insertion).

pBac_STBL:

A 2.7 kb EcoRI-BfrI fragment (both restriction sites filled in by Klenowreaction) from pSL-3×P3-DsRed-FRT-pBacR2-FRT is cloned intopBac-3×P3-EYFPaf (see WO 01/12667, the contents of which areincorporated herein by reference) previously cut with BglII (Klenowfill-in reaction). The insertion with an opposite orientation of theDsRed- and EYFP-ORFs is chosen. This final plasmid contains piggyBacR2in opposite orientation to piggyBacR1 (FIG. 6).

phspBac Transposase Helper Plasmid:

For germline transformation experiments, the helper phspBac is used (seePCT WO 01/14537, the contents of which are incorporated herein byreference).

Experimental Steps of the Transgene Immobilization Process (FIG. 4 andFIG. 6)

a) Germline Transformation of pBac_STBL (Step 1 in FIG. 4 and FIG. 6)

DNA-sequences included in the plasmid pBac_STBL within the ends ofpiggyBacL1 and piggyBacR1 are integrated into the Drosophila genome bypiggyBac-mediated germline transformation (see U.S. Pat. No. 6,218,185and WO 01/14537, the contents of which are incorporated herein byreference). Similar constructs incorporating genes-of-interests insertedat the unique cloning sites would be treated in the same way.

b) FLP Recombinase Induced Inversion (Step 2 in FIG. 4 and FIG. 6)

Genomic integrations of the pBac_STBL transgene are identifiable by bothEYFP and DsRed eye fluorescence (see WO 01/12667, the contents of whichare incorporated herein by reference). Following the identification oftransgenic founder individuals (and to establish Drosophila strainscarrying the transgene in the homozygous state), the inversion of thepiggyBacR2 sequence is carried out. This is performed by crossing in thestrain beta2t-FLP that expresses FLP-recombinase during spermatogenesis.Alternatives of step 2 in FIG. 6 include crossing in hsp70-FLP andhsFLP-strains, respectively, or microinjection of a FLP-recombinaseencoding plasmid, e.g. pKhsp82-FLP (into preblastoderm embryos ofhomozygous transgenic pBac_STBL lines). Though the inversion eventcannot be detected by the marker genes included into pBac_STBL, astatistical equilibrium of original and inverted orientation of thepiggyBacR1 sequence can be assumed. Thus, the inversion process isdetected by testing several independent sublines by sequencing of vectorPCR products to identify sublines having undergone piggyBacR1 inversion.

c) PiggyBac Transposase Induced Deletion (Step 3 in FIG. 4 and FIG. 6)

Strains with inverted piggyBacR2 sequence are crossed to piggyBactransposase expressing strains (referred to as jumpstarter). Differentlines of the Drosophila strain Her{3×P3-ECFP, alphaltub-piggyBacK10} areavailable for this step. Progeny from this cross expressing bothEYFP/DsReD (indicating the presence of pBac_STBL) and ECFP (indicatingthe presence of the jumpstarter) are crossed out in single male setups.

d) Identification of Immobilized Transgene DNA

ECFP⁻ progeny (selection against the jumpstarter) of single malecrossings are analyzed for both the presence of EYFP fluorescence andthe absence of DsRed fluorescence. Individuals putatively containing atransposon deletion event should show EYFP but absence of DsRedfluorescence and can be analyzed further. By inverse PCR, the transposondeletion can be molecularly confirmed and stability of the potentiallyimmobilized transgene insertion can be assessed by challenging thetransgene insertion with piggyBac transposase.

Embodiment 3 RMCE with Subsequent Transposon Deletion

The RMCE-acceptor plasmid, pBac{3×P3-FRT-ECFP-linotte-FRT3} (FIG. 8), isa piggyBac-based transformation vector that was provided additionallywith a DNA exchange cassette. This cassette consists of twoheterospecific FRT sites (referred to as FRT and FRT3 equivalent to Fand F3 (published in European Patent No. EP 0 939 120 A1, the contentsof which are incorporated herein by reference)) in parallel orientation.

European Patent No. EP 0 939 120 A1 (see page 2, line 50 to page 3, line6) teaches the technology of the RMCE reaction:

-   -   “Recombinases such as FLP and Cre have emerged as powerful tools        to manipulate the eucaryotic genome (Kilby, N. J., Snaith, M.        R., Murray, J. A. H. (1993). Site-specific recombinases: tools        for genome engineering. Trends Genet. 9, 413-421, and Sauer B.        (1994). Site-specific recombination: developments and        applications. Curr. Opin. Biotechnol. 5, 521-527, the contents        of which are incorporated by reference herein). These enzymes        mediate a recombination between two copies of their target        sequence and have mainly been used for deletions. We show here        that FLP-RMCE can be applied to introduce secondary mutations at        a locus which has been previously tagged by a positive/negative        selectable marker, and that these secondary mutations can be        produced without depending on a selectable marker on the        incoming DNA. FLP-RMCE utilizes a set of two 48 bp. FLP target        sites, in this case wild type (F) and F3, a mutant that was        derived from a systematic mutagenesis of the 8 bp spacer        localized between the FLP binding elements (see Schlake, T.,        Bode, J. (1994). Use of mutated FLP recognition target (FRT)        sites for the exchange of expression cassettes at defined        chromosomal loci. Biochemistry 33, 12746-12751, the contents of        which are incorporated by reference herein). FLP effects        recombination between the F3/F3 couple which is as efficient as        between the wild type sites (F/F) but it does not catalyze        recombination between a F/F3 pair (Seibler J., Bode J. (1997).        Double-reciprocal crossover mediated by FLP-recombinase: a        concept and an assay. Biochemistry 36, 1740-1747, the contents        of which are incorporated by reference herein). Thereby FLP-RMCE        enables the specific exchange of an expression cassette in the        genome which is flanked by a F3-site on one end and a F-site on        the other for an analogous cassette comprising virtually any        sequence which is provided on a plasmid in a single step without        the need of introducing a positive selectable marker. Nothing        else in the genome is altered and no plasmid sequences are        inserted. In contrast to approaches using a single recombination        site the targeting product is stable even under the permanent        influence of the recombinase unless it is exposed to an exchange        plasmid (Seibler J., Bode J. (1997). Double-reciprocal crossover        mediated by FLP-recombinase: a concept and an assay.        Biochemistry 36, 1740-1747, the contents of which are        incorporated by reference herein). The system can be used to        analyze the function of either a gene product or of regulatory        sequences in ES-cells or of the derived transgenic mice.”        (citations added)

In the present invention, FRT and FRT3 flank the ECFP open reading frameand a “homing sequence”. As a “homing sequence”, the 1.6 kb HindIIIfragment of the Drosophila linotte locus was chosen (see Taillebourg, E.& Dura, J. M. (1999). A novel mechanism for P element homing inDrosophila. Proc. Natl. Acad. Sci. USA 96, 6856-6861, the contents ofwhich are incorporated herein by reference. This particular sequence hasbeen described to act as “bait” for homing of identical/homologous DNAsequences by a process called “para-homologous pairing”. We have shownpreviously that the positioning of the FRT site between the 3×P3promoter and the start codon of the ECFP open reading frame does notinterfere with expression of the 3×P3-ECFP gene (see PCT WO 01/12667,the contents of which are incorporated herein by reference). The RMCEdonor plasmid, pSL-FRT-EYFP-pBacR2-3×P3-DsRed-linotte-FRT3 (FIG. 10),contains the DNA cassette to be recombined in. The donor cassettecomprises the two heterospecific FRT sites (FRT and FRT3) flanking theEYFP open reading frame (promoter-free), a piggyBacR2 3′-half sidesequence, the transformation marker gene 3×P3-DsRed and the homingsequence from the linotte locus (identical to the linotte sequence inthe RMCE acceptor). The RMCE donor plasmid is a derivative of theplasmid pSLfa1180fa (see Patent Cooperation Treaty PCT WO 01/12667 A1),which does not contain any transposon sequences. AscI/FseI cloning siteshave been incorporated to ease the insertion of gene(s)-of-interestupstream of the piggyBacR2 sequence.

In the following, the details of the RMCE plasmids construction startingfrom plasmid already published are disclosed:

Construction of the RMCE Acceptor Plasmid (FIG. 8):

pSL-3×P3-FRT-ECFPaf:

A 90 bp SalI-Asp718 fragment from the plasmid pSL>AB> containing the FRTsequence was cloned into the plasmid pSL-3×P3-ECFPaf (see PatentCooperation Treaty PCT WO 01/12667, the contents of which areincorporated herein by reference) previously digested with SalI-Asp718.The FRT sequence corresponds to the substrate of the FLP recombinase:

TT GAAGTTCCTATTC C GAAGTTCCTATTC TCTAGAAA GTATAGGAACTTCAGAGCGCTTTTGAAGCTpBac{3×P3-FRT-ECFPaf}:

A 1.3 kb EcoRI-(blunted by Klenow fill in reaction)-NruI fragment fromthe plasmid pSL-3×P3-FRT-ECFPaf was cloned into the plasmid p3E1.2previously digested with HpaI.

pBac{3×P3-FRT-ECFP-linotte-FRT3}, Final RMCE Acceptor Plasmid:

The plasmid pBac{3×P3-FRT-ECFPaf} was digested with AscI-BglII, and thefollowing sequences were cloned into the linearized vector:

i) the AscI-Asp718 cut PCR amplification product of the 1.6 kb HindIIIgenomic linotte fragment. As a template, genomic DNA of Drosophilamelanogaster, strain OregonR, was chosen and as primers:

CH_lioFwd (5′-TTGGCGCGCCAAAAGCTTCTGTCTCTCTTTCTG-3′) and CH_lioRev(5′-CGGGGTACCCCAAGCTTATTAGAGTAGTATTCTTC-3′) and

ii.) the Asp718-BglII cut PCR amplification product of the FRT3 sequence(mutagenic PCR). As a template, the plasmid PSL>AB> was chosen and asprimers:

CH_F3Fwd (5′-TTGGCGCGCCAAGGGGTACCCGGGGATCTTG-3′) und

CH_F3Rev (5′-CGCTCGAGCGGAAGATCTGAAGTTCCTATACTATTTGAAGAATAG-3′).

The FRT3 sequence corresponds to the F3 sequence (European Patent No. EP0 939 120 A1):

TT GAAGTTCCTATTC C GAAGTTCCTATTC TtcAaAtA GTATAGGAACTTC AGAGCGC

The diagram of this final RMCE acceptor vector is shown in FIG. 8.

Construction of the RMCE Donor Plasmid (FIG. 10)

pSL-3×P3-FRT-EYFPaf:

Construction was analogous to pSL-3×P3-FRT-ECFPaf, but into the plasmidpSL-3×P3-EYFPaf (see WO 01/12667, the contents of which are incorporatedherein by reference).

pSL-FRT-EYFPaf:

The 3×P3 promoter sequence was deleted from the plasmidpSL-3×P3-FRT-EYFPaf by digestion with EcoRI-BamHI, filling-in by Klenowenzyme reaction and finally religating the blunted plasmid.

pSL-FRT-EYFP-linotte-FRT3:

A 1.7 kb AscI-BglII (both sites blunted by Klenow fill-in reaction)fragment from pBac{3×P3-FRT-ECFP-linotte-FRT3} was cloned into theplasmid pSL-FRT-EYFPaf previously digested with NruI. The orientationwith maximal distance of the FRT and FRT3 sites was chosen.

pBac{3×P3-DsRedaf}:

A 1.2 kb EcoRI (site blunted by Klenow fill-in reaction)-NruI fragmentfrom the plasmid pSL-3×P3-DsRedaf was cloned into the plasmid p3E1.2(see U.S. Pat. No. 6,218,185, the contents of which are hereinincorporated by reference) previously digested with BglII-(site bluntedby Klenow fill-in reaction)-HpaI.

pSL-FRT-EYFP-linotte-FRT3-3×P3-DsRed:

A 1.25 kb EcoRI-(site blunted by Klenow fill-in reaction)-NruI fragmentfrom pSL-3×P3-DsRedaf was cloned into the plasmidpSL-FRT-EYFP-linotte-FRT3 previously digested with SpeI (site blunted byKlenow fill-in reaction).

pSL-FRT-EYFP-pBacR-3×P3-DsRed-linotte-FRT3, Final RMCE Donor Plasmid:

A 2.5 kb AscI-(site blunted by Klenow fill-in reaction)-EcoRV fragmentfrom pBac{3×P3-DsRedaf} was cloned into the plasmidpSL-FRT-EYFP-linotte-FRT3 previously cut with EcoRI (site blunted byKlenow fill-in reaction).

The diagram of this final RMCE acceptor vector is shown in FIG. 10.

FLP Recombinase Plasmid Source: pKhsp82-FLP:

A 2.2 kb Asp718-XbaI (sites blunted by Klenow fill-in reaction) fragmentfrom the plasmid pFL124 containing the FLP recombinase ORF and the 3′transcriptional terminator from the adh gene was cloned into the plasmidpKhsp82) previously cut with BamHI (site blunted by Klenow fill-inreaction).

phspBac Transposase Helper Plasmid:

For germ-line transformation experiments, the helper phspBac was used(see PCT WO 01/14537 A1).

DNA Cassette Exchange by RMCE is Highly Efficient in Drosophilamelanogaster

Practical application of RMCE-based gene targeting and germlinetransformation (e.g. for the purpose of immobilizing transgenes) willdepend strongly on the efficiency of the DNA cassette exchange. Thisefficiency should be in the range observed with conventionaltransposon-mediated germline transformation systems that allow theisolation of several transgenic founder individuals among 1,000-10,000progeny screened. Previous experiments involving DNA cassette exchangehave been performed only using cell culture and stringent selectionconditions. Therefore the efficiency of such a system in an invertebrateorganism such as Drosophila is hard to predict. Hence, a pilotexperiment was performed. An intermediate of the RMCE donor plasmid,pSL-FRT-EYFP-linotte-FRT3 and the FLP recombinase expression vectorpKhsp82-FLP were co-injected into pre-blastoderm embryos of a Drosophilamelanogaster acceptor strain. These embryos carry the RMCE acceptortransgene vector (FIG. 8) integrated by piggyBac-mediated germ-linetransformation, in a homozygous state. The final concentration of theplasmids in the injection mix was 500 ng/μl (RMCE donor plasmid) and 300ng/μl (pKhsp82-FLP). Altogether, around 3,000 Drosophila embryos wereinjected, corresponding to ten times the number necessary for aconventional piggyBac-mediated germ-line transformation. Successfulexchange of the acceptor by the donor cassette was indicated by thechange in the eye fluorescence from ECFP to EYFP (in F1 individuals).Results documenting the frequency of such exchange events are given inTable 1:

TABLE 1 Results of the RMCE experiment in Drosophila with the donorplasmid pSL-FRT-EYFP-linotte-FRT3. Acceptor lines (II: second, III:third chromosomal homozygous, ECFP fluorescence) used formicroinjection, number of injected embryos, male and fertile maleinjection survivors and the number of vials containing EYFP-positiveprogeny are given. Male Fertile Male Vials Acceptor Injected Injectionwith EYFP-pos. and Line Embryos Survivors Inj. Surv. ECFP-neg. progenyM4.II ECFP 750 121 70 22 M7.III ECFP 750 138 72 17 M8.II ECFP 600 68 5412 M9.II ECFP 750 123 109 27

EYFP-positive founder males resulting from targeting events were bred tohomozygosity and established as stocks (referred to as “M4.II EYFP”,“M7.III EYFP”, “M8.II EYFP” and “M9.II EYFP”, respectively). Segregationanalysis (genetic mapping of transgene integrations) indicated for allfour lines that the chromosomal localization of the donor and acceptortransgene is identical.

We define the DNA cassette exchange frequency as a percentage of fertileF₁ vials producing EYFP-positive progeny. With this definition, thefrequency of RMCE events is 25% on average corresponding well to thegerm-line transformation frequency usually observed with piggyBac,Hermes or Minos-based vectors in Drosophila). This experimentdemonstrates that, with the particular design of RMCE-vectors, theprocess of cassette exchange is highly efficient in an invertebrateorganism such as Drosophila.

Molecular Characterization of RMCE Events and Integration Site Analysis

a) Genomic Integration Site of Donor and Acceptor Transgenes

The exchange of eye fluorescence from ECFP to EYFP suggests that thedonor cassette (carrying the promotor-free eyfp gene) integrated at thelocus of the acceptor transgene (providing the 3×P3 promoter).Therefore, the genomic integration sites of the acceptor transgene inthe acceptor line and of the donor transgene in the corresponding donorline should be identical. To identify genomic integration sites, inversePCR experiments were carried out for acceptor and donor Drosophilalines. To recover DNA sequences flanking piggybac insertions, inversePCR was performed. The purified fragments were directly sequenced forthe 5′ junction with primer CH_PLSeq 5′-CGGCGACTGAGATGTCC-3′. Theobtained sequences were used in BLAST searches against the DrosophilaGenome Sequence Database. For the 5′ junction, genomic DNA sequenceidentity could be confirmed for three acceptor/donor pairs (Table 2).

TABLE 2 Genomic integration sites of the acceptor transgene pBac{3xP3-FRT-ECFP-linotte-FRT3} in four Drosophila lines used for RMCEtargeting. Sequence numbers and nucleotide positions refer to theRelease 3 sequence of the Drosophila Genome Sequence Database. IdenticalLocation for corre- of insert sponding Acceptor Chromosome genomic donorline arm scaffold position line? M4.II ECFP 2L AE003662.3 204692 yes(M4.II EYFP) M7.III ECFP 3L AE003558.3 171057 yes (M7.III EYFP) M8.IIECFP 2L AE003618.2 15414 yes (M8.II EYFP) M9.II ECFP 2L AE003662.3 15805nd.For three corresponding RMCE donor lines, integration sites could beconfirmed to be identical. nd.: not determined

Interestingly, the acceptor line M9.II ECFP was found to carry theacceptor transgene integrated at the Drosophila-endogenous linotte locus(integration position corresponds to bp 1185). This suggests that“para-homologous pairing” of the linotte sequences included in theacceptor plasmid to the homologous genomic sequence occurred, furtherverifying the homing phenomenon.

b) Southern Analysis

To further verify at the molecular level that the donor transgenetargeted the acceptor locus via an RMCE mechanism, Southern analysis ongenomic DNA of the four acceptor and the four donor lines was performed.PstI was chosen as an indicative restriction digest and a probehybridizing to gfp-based transformation marker genes (hybridizing toboth ECFP and EYFP) was selected (FIG. 9). Only one strong hybridizationsignal was present in all acceptor lines which is consistent with asingle integration of the acceptor transgene. The expected pattern ofDNA-DNA hybridization, 2.4 kb for the acceptor transgene and 1.6 kb forthe donor transgene, was detected for all four lines for each transgene(FIG. 9). Additionally, a −6 kb hybridization signal was detected onlyin RMCE donor lines. As this signal might indicate the presence of thecomplete donor vector, further Southern experiments (using probesagainst the pUC plasmid backbone sequences) were carried out. Thepresence of pUC sequence in the donor lines could be confirmed (data notshown) pointing toward an integration of the entire donor vector in thefour donor lines analyzed.

In summary, three lines of evidence let us infer that targeting of theRMCE acceptor locus by the RMCE donor vector took place: i) the exchangein eye color fluorescence from ECFP (acceptor) to EYFP (donor), ii) theidentity of genomic DNA sequence flanking the piggyBac transgeneintegration in corresponding acceptor and donor lines, and iii) DNAhybridization signals in accordance with expectations for the exchangeof the ecfp to the eyfp open reading frame.

Recombination Occurs by Cassette Exchange Via FRT and FRT3

The recombinase-mediated cassette exchange mechanism requires a doublerecombination event (see European Patent No. EP 0 939 120, the contentsof which are incorporated herein by reference). Because the Southernanalysis suggests that in the pilot RMCE experiments singlerecombination events caused integration of the entire donor plasmid, weanalyzed in more detail whether the RMCE mechanism, which has not beenestablished for an invertebrate organism, can occur in Drosophila. Tothis end, we modified the donor construct to include a 3×P3-DsRed markergene downstream to the FRT3 sequence(pSL-FRT-EYFP-linotte-FRT3-3×P3-DsRed). This vector configuration allowsthe separation of RMCE events:

-   1) double cross-over via FRT and FRT3 sites resulting in ECFP to    EYFP eye fluorescence exchange-   2) single recombination events (via FRT site) resulting in ECFP to    EYFP and DsRed eye fluorescence exchange-   3) single recombination events (via FRT3 site) resulting in ECFP to    DsRed (and ECFP) eye fluorescence exchange

For the targeting experiment, the acceptor line M4.II ECFP (Table 1) wasselected for further testing. F1 individuals with ECFP to EYFP exchangeindicating targeting were observed at a frequency of 13.1%:

Embryos injected: 750 single G0 male founders: 109 Fertile G0 malefounders: 84 Setups producing EYFP-fluorescing F1 progeny: 11

The eleven setups yielding EYFP-fluorescing individuals were analyzedfor the occurrence of double and single recombination events (Table 3).

TABLE 3 Phenotypic analysis of F1 progeny from G0 male founders of theacceptor line M4.II ECFP injected with the donorpSL-FRT-EYFP-linotte-FRT3-3xP3-DsRed. Double and single recombinationevents are indicated by differential analysis of eye fluorescence forECFP, EYFP and DsRed. Phenotype of individual flies double recombinationsingle FRT rec.single FRT3 rec. EYFP⁺ DsRed⁺DsRed⁺, Setup# (DsRed⁻,EYFP⁺, ECFP⁺ Stocks ECFP⁻) (ECFP⁻) (EYFP⁻) established 1 1 1 0 2 1 0 0R1 3 2 6 0 4 4 0 0 R2 5 3 0 0 R3 6 3 0 0 R4 7 1 10 0 R5 (EYFP⁺, DsRed⁺)8 13 26 0 R3 (EYFP⁺, DsRed⁺) 9 1 2 0 10 11 3 0 11 1 0 0

Five out of eleven setups produced progeny showing EYFP but lackingDsRed (and ECFP) fluorescence. This phenotype is consistent withtargeting via double recombination with only sequences between FRT andFRT3 being exchanged. However, single recombination events via FRT werealso observed, in contrast to no single recombinations via FRT3. Theresults indicate that recombinase mediated cassette exchange ismechanistically feasible in an invertebrate organism (the vinegar flyDrosophila melanogaster) and, by applying a simple eye fluorescencemarker scheme, double recombination events can be selected for.

Experimental Steps of the Transgene Immobilization Process (FIG. 5 andFIG. 7)

The previous results demonstrate that recombinase mediated targeting ofgenomic DNA loci is possible in an invertebrate organism likeDrosophila. As depicted in FIG. 5, the RMCE strategy can be furtheremployed for the purpose of post-transformational transgeneimmobilization. The general procedure consists of two steps. In thefirst step, a transformation vector containing the gene of interest, atransposon half-side (TransposonR2 in FIG. 5) and an additional markergene is used as the RMCE donor to target the RMCE acceptor line (i.e.RMCE acceptor vector (FIG. 8) genomically integrated). By a single ordouble recombination event, an ‘internal’ piggyBac transposon comprisingboth half-sides (piggyBacL1 and piggyBacR2 in FIG. 5) is reconstituted.In a second step, transposase activity is introduced to remobilize the‘internal’ transposon by selecting for individuals lacking theadditional marker gene as demonstrated in embodiment 1.

In the following section we provide data that prove this principle:

Step 1: Targeted DNA Cassette Exchange (RMCE, Step 1 in FIG. 5 and FIG.7)

The final donor plasmid, pSL-FRT-EYFP-pBacR2-3×P3-DsRed-linotte-FRT3(FIG. 10, in the following referred to as “final RMCE donor”) contains,in-between the FRT and FRT3 sites, a cassette with: (i) a promotor-freeeyfp ORF, (ii) the piggyBacR2 (3′ end) transposon sequence, (iii) thetransformation marker 3×P3-DsRed, and (iv) the homing sequence from theDrosophila linotte locus (see Taillebourg, E. & Dura, J. M. (1999). Anovel mechanism for P element homing in Drosophila. Proc. Natl. Acad.Sci. USA 96, 6856-6861, the contents of which are incorporated herein byreference). Derivatives of the final RMCE donor vector carryingadditional DNA sequences (genes-of-interest) can be constructed byinsertion into the unique AscI and FseI cloning sites which are locatedupstream of the piggyBacR2 transposon sequence (FIG. 10).

Microinjection of the final RMCE donor was carried out using theDrosophila acceptor line M4.II ECFP (Table 2). This line carries theacceptor transgene pBac{3×P3-FRT-ECFP-linotte-FRT3} in the homozygousstate. Embryos were injected under the conditions described previously.Single G0 founder males were crossed out and progeny (generation F1)were screened for the presence of both EYFP fluorescence and DsRedfluorescence (see FIG. 7). Targeting (i.e. individuals with ECFP to EYFPexchange) were observed at a frequency of 22.2%.

Embryos injected: 750 single G0 male founders: 178 Fertile G0 malefounders: 158 Setups producing EYFP and DsRed fluorescing F1 progeny: 34

In total, 91 female and 62 male individuals were obtained whichconsistently showed an EYFP and DsRed eye fluorescence phenotype.Moreover, in these individuals ECFP fluorescence was absent as expectedfor recombination events. Though the exact mechanism (single versusdouble recombination) was not investigated for individuals from thistargeting experiment, the previous pilot experiments suggest asignificant fraction of double recombination events resulting fromcassette exchange via FRT and FRT3 sites.

The results confirm a high efficiency of the gene targeting systemdisclosed in this embodiment, which is comparable to ‘conventional’transposon-mediated germ-line transformation, at least for the vinegarfly Drosophila. In particular, the efficiency did not decreasesignificantly due to the interruption of the linotte sequence in thefinal donor plasmid or the increased size (2.6 kb compared to previous“pilot” donor vector) of the final donor plasmid (FIG. 10). Thissuggests that recombinants can also be generated with derivatives of thefinal donor plasmid carrying additional gene(s)-of-interest.

Step 2: PiggyBac Transposase Induced Transposon Deletion of a TargetedVector (Step 2 in FIG. 5 and FIG. 7).

Successful re-mobilization of the reconstituted piggyBac transposon isindicated by loss of DsRed fluorescence. Progeny lacking the sequencebetween piggyBacR2 and piggyBacL1 exclusively express EYFP fluorescence(see FIG. 7).

To examine whether the reconstituted internal piggyBac transposon vectorcan be re-mobilized by piggyBac transposase activity, individuals ofgeneration F1 with EYFP and DsRed eye fluorescence were crossed to thefollowing piggyBac-expressing jumpstarter lines:

-   (1) line Her{3×P3-ECFP; αtub-piggyBac} M6.II, referred to as “HerM6”-   (2) line Her{3×P3-ECFP; αtub-piggyBac} M10.III, referred to as    “HerM10”-   (3) line Mi{3×P3-DsRed; hsp70-piggyBac} M5.II, referred to as “MiM5”

Progeny (generation F2) carrying both the final RMCE donor and thejumpstarter transgenes were crossed individually to non-transgenicDrosophila and progeny from these crosses (generation F3) were analyzedfor the presence of individuals carrying EYFP but lacking DsRed eyefluorescence (Table 4).

TABLE 4 Phenotypic analysis for piggyBac transposon remobilizationevents. Progeny from single crosses of males carrying both final RMCEdonor and jumpstarter transgenes (Js) to non- transgenic Drosophilavirgin females were analyzed for individuals showing EYFP eyefluorescence but lacking DsRed eye fluorescence. Js HerM6 HerM10 MiM5Setup EYFP⁺ DsRed⁻ EYFP⁺ DsRed⁻ EYFP⁺ DsRed⁻  1 73 0 62 0 38 32  2 67 057 1 42 0  3 47 0 48 0 56 1  4 53 0 52 0 68 3  5 36 0 34 0 48 5  6 61 148 1 37 0  7 50 1 55 0 38 1  8 40 0 52 0 71 5  9 39 0 55 0 41 0 10 86 043 0 72 2 11 53 0 40 0 49 0 12 57 0 71 0 30 0 13 17 0 52 0 46 0 14 58 166 0 48 1 15 65 2 56 0 41 0 16 54 2 55 0 54 0 17 55 2 51 0 53 0 18 54 043 1 66 2 19 63 1 18 0 56 1 20 78 0 38 1 63 1 Sum: 1106 10 996 4 1017 25Such a phenotype is consistent with a deletion of the internallyreconstituted piggyBac transposon (FIG. 7).

Depending on the jumpstarter line employed, the frequency ofremobilization ranged from 0.4% (HerM10) to 2.5% (MiM5). This indicatesthat the reconstituted internal piggyBac transposon vector can beremobilized efficiently, and the combination of different fluorescencemarkers allows the straightforward identification of remobilizationevents. Finally, the physical deletion of the reconstituted piggyBactransposon could be verified at a molecular level by PCR analysis (FIG.9): Utilizing a primer pair binding to genomic region flanking to theRMCE acceptor transgenic line M4.II (primer M4.II Rev) and to piggyBacL1sequences (primer pBL-R), the deletion of piggyBacL1 could be confirmed(compare PCR amplification products for acceptor line M4.II andimmobilized lines #7 and #8 in FIG. 9). Moreover, utilizing a primerpair binding to genomic region flanking to the RMCE acceptor transgenicline M4.II (primer M4.II Rev) and to the linotte sequence (primerlioFwd) the truncation of the immobilized transgene could be confirmed(FIG. 9). The piggyBac remobilization event can be further confirmed byDNA sequencing over the genomic DNA to transgene DNA junction.

In conclusion, our data provide a proof-of-principle for the strategy oftransgene immobilization by “RMCE with subsequent transposon deletion”in an invertebrate organism (Drosophila melanogaster).

ADVANTAGES OF THE INVENTION OVER THE PRIOR ART

The major advantage of the novel transformation systems disclosed inthis patent application is the possibility to physically deletetransposon DNA following the germ-line transformation process, inaddition to targeting transgene integrations into predefined targetsites. In this way, transposase-mediated mobilization orcross-mobilization of the genes-of-interest are excluded mechanisticallyand random genomic integrations are eliminated. In contrast toconventional germ-line transformation technology, our systems provideenhanced stability to the transgene insertion. Furthermore, DNAsequences required for the modification (e.g. transformation markergenes, transposase or recombinase target sites) are, to a large extent,removed from the genome after the final experimental step (step 2 inFIG. 1, step 3 in FIG. 4 and step 2 in FIG. 5). The final transgeneinsertion does not contain DNA sequences encoding complete target sitesfor the recombinases or transposases employed during the process,thereby eliminating the possibility for instability generated by theseprocesses.

The RMCE technology, which is disclosed in this patent application forinvertebrate organisms (exemplified in Drosophila melanogaster)represents an extremely versatile tool with application potential farbeyond the goal of transgene immobilization. RMCE makes possible thetargeted integration of DNA cassettes into a specific genomic DNA locus.This locus is pre-defined by the integration of the RMCE acceptorplasmid and can be characterized prior to a targeting experiment. Inaddition to the expected expression properties of the transgenes(including strength of expression, stage-specificity,tissue-specificity, and sex-specificity), the genomic environment of thetransgene integration can have a significant effect on the level andtissue-specificity of expression. Therefore, suitable loci forintegrations can be pre-selected before performing a gene targetingexperiment according to the requirements specific for the experimentalsetup, and in addition, host strains with optimal fitness may beselected. Moreover, multiple cassette exchange reactions can beperformed in a repetitive way, i.e. an acceptor cassette in a particularinvertebrate strain with a specific genetic makeup can be repetitivelyexchanged by multiple donor cassettes. Furthermore, several differenttransgenes can be placed exactly at the same genomic locus. This allowsfor the first time the ability to eliminate genomic positional effectsand to comparatively study the biological effects of differenttransgenes.

The particular embodiments of the invention are highly flexible. Thefunctionality of systems disclosed is neither dependent on theparticular transposable elements used in the embodiments, nor on theparticular transformation marker genes used in the embodiments, nor onthe particular site-specific recombination system used in theembodiments, nor on the particular homing sequence used in embodiment 3.Finally, all embodiments have broad general application potential invertebrate and invertebrate organisms that are subject totransposon-mediated transformation or recombinase-mediatedrecombination, and fluorescent protein marking systems.

1: A method for producing a heritable integration of a transgene withina genome of a somatic or germ line cell of an invertebrate organism, themethod comprising: providing a first DNA cassette within said genome,wherein said first cassette comprises a first flanking transposon halfside, a second flanking transposon half side, and an internal transposonhalf side, wherein said internal transposon half side and said firstflanking transposon half side form a pair of excisable transposonhalf-sides, and wherein said first cassette further comprises saidtransgene in-between the internal transposon half side and said secondflanking transposon half side; and mobilizing said excisable transposonhalf-sides. 2: The method of claim 1, wherein said internal transposonhalf side and said second flanking transposon half side are TransposonLhalf sides, and wherein said first flanking transposon half side is aTransposonR half side. 3: The method of claim 1, wherein said internaltransposon half side and said second flanking transposon half side areTransposonR half sides, and wherein said first flanking transposon halfside is a TransposonL half side. 4: The method of claim 1, wherein saidexcisable transposon half-sides and corresponding transposase enzyme arefrom a transposable element, wherein said transposable element hasterminal inverted sequences, and wherein said transposable elementtransposes via a DNA-mediated process. 5: The method of claim 1, whereinsaid first DNA cassette further comprises a first selectable marker genelocated between said internal transposon half side and said firstflanking transposon half side, and a second selectable marker genelocated between said internal transposon half side and said secondflanking transposon half side, and wherein said first and secondselectable marker genes are phenotypically distinguishable. 6: Themethod of claim 5, wherein said first and second marker genes are, ineither order, any combination of marker genes producing distinguishablefluorescent or other visible dominant phenotypes. 7: The method of claim5 wherein said first and second marker genes are, in either order, acombination of the transformation marker genes PUbDsRed1 and 3×P3-ECFP.8: The method of claim 1, wherein said internal transposon half side isprovided in reverse orientation, wherein said excisable transposon isformed by inversion of said internal transposon half side relative tosaid first flanking transposon half side, wherein said internaltransposon half side further comprises flanking recombinase sites, andwherein said inversion is catalyzed by a site-specific recombinase. 9:The method of claim 8, wherein said recombinase sites are FRT sites inopposite or reverse orientation. 10: The method of claim 1, wherein saidexcisable transposon is mobilized by a source of transposasecorresponding to said excisable transposon to render the remaininggenomic DNA immobilizable. 11-22. (canceled) 23: An invertebrateorganism comprising the heritable transgene produced according toclaim
 1. 24. (canceled) 25: A method for producing a heritableintegration of a transgene within a genome of a somatic or germ linecell of an organism, the method comprising: providing a first DNAcassette within said genome, wherein said first cassette comprises afirst flanking transposon half side, a second flanking transposon halfside, and an internal transposon half side, wherein said internaltransposon half side and said first flanking transposon half side form apair of excisable transposon half-sides, and wherein said first cassettefurther comprises said transgene in-between the internal transposon halfside and said second flanking transposon half side; and mobilizing saidexcisable transposon half-sides. 26: The method of claim 25, whereinsaid internal transposon half side and said second flanking transposonhalf side are TransposonL half sides, and wherein said first flankingtransposon half side is a TransposonR half side. 27: The method of claim25, wherein said internal transposon half side and said second flankingtransposon half side are TransposonR half sides, and wherein said firstflanking transposon half side is a TransposonL half side. 28: The methodof claim 25, wherein said excisable transposon half-sides andcorresponding transposase enzyme are from a transposable element,wherein said transposable element has terminal inverted sequences, andwherein said transposable element transposes via a DNA-mediated process.29: The method of claim 25, wherein said first DNA cassette furthercomprises a first selectable marker gene located between said internaltransposon half side and said first flanking transposon half side, and asecond selectable marker gene located between said internal transposonhalf side and said second flanking transposon half side, and whereinsaid first and second selectable marker genes are phenotypicallydistinguishable. 30: The method of claim 29, wherein said first andsecond marker genes are, in either order, any combination of markergenes producing distinguishable fluorescent or other visible dominantphenotypes. 31: The method of claim 29, wherein said first and secondmarker genes are, in either order, a combination of the transformationmarker genes PUbDsRed1 and 3×P3-ECFP. 32: The method of claim 25,wherein said internal transposon half side is provided in reverseorientation, wherein said excisable transposon is formed by inversion ofsaid internal transposon half side relative to said first flankingtransposon half side, wherein said internal transposon half side furthercomprises flanking recombinase sites, and wherein said inversion iscatalyzed by a site-specific recombinase. 33: The method of claim 32,wherein said recombinase sites are FRT sites in opposite or reverseorientation. 34: The method of claim 25, wherein said excisabletransposon is mobilized by a source of transposase corresponding to saidexcisable transposon to render the remaining genomic DNA immobilizable.35-46. (canceled) 47: An organism comprising the heritable transgeneproduced according to claim
 25. 48. (canceled)